Concatenation cDNA sequencing for transcriptome analysis

We describe a high-throughput cDNA sequencing pipeline (http://www.hgsc.bcm.tmc.edu/projects/cdna) built in response to the emerging need for rapid sequencing of large cDNA collections. Using this strategy cDNA inserts are purified and joined through concatenation into large molecules. These 'pseudo-BACs' are subjected to random shotgun sequencing whereby the majority of cDNA inserts in the pool are sequenced. Using this concatenation cDNA sequencing platform, we have contributed more than 13000 full-length cDNA sequences from human and mouse to the Mammalian Gene Collection (MGC).     

Scalable transcriptome preparation for massive parallel sequencing

Background

The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity.

Methodology

In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation.

Conclusion/Significance

The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods.